In the course of performing their normal physiological functions, many types of cells, including bacterial cells and those in specialized mammalian tissues such as the liver and kidney, transport a variety of organic molecules across their cell membranes. For example, cells in the proximal tubule of the kidney transport glucose, amino acids, and uric acid across their membranes, and work to eliminate various drugs and toxic substances from the body. All of these molecules are transported across the cell membranes by specialized cellular transporters.
Recently, genes encoding several putative transporters have been identified. These molecules include OCT-1 (organic cation transporter; Grundemann et al., Nature 372:549-552, 1994), OCT-2 (Okuda et al., Biochem. Biophys. Res. Comm. 224:500-507, 1996), NLT (novel liver-specific transporter; Simonson et al., J. Cell Sci. 107:1067-1072, 1994), and NKT (novel kidney-specific transporter; Lopez-Nieto et al., J. Biol. Chem. 272:6471-6478, 1997). While the sequences of these transporters are not highly conserved (at the amino acid level, OCT-1 and NLT are only 30% and 35% identical to NKT, respectively), they do exhibit similar transmembrane (TM) domain hydropathy profiles.
The invention described herein relates to the discovery and characterization of oct-3, a gene encoding a protein that transports molecules across the plasma membranes of biological cells. OCT-3 is highly expressed in the neuronal cells of the brain, including, e.g., cells in the cortex and in the hypothalamus. Accordingly, altering the expression or activity of OCT-3 (e.g., with small molecules, antisense molecules, or neutralizing antibodies) can alter the concentration of molecules (such as neurotransmitters) that are present within the cell or in the extracellular spaces around the cell (i.e., on either side of the plasma membrane). Altering the concentrations of these molecules in patients afflicted with certain conditions, including neurodegenerative diseases, behavioral disorders, eating or sleep disorders, and pain responses (e.g., elicited during various forms of tissue injury or in association with musculoskeletal disorders), can provide relief from the symptoms associated with these conditions.
More specifically, the invention features an isolated nucleic acid molecule (i.e., a nucleic acid molecule that is separated from the 5xe2x80x2 and 3xe2x80x2 coding sequences with which it is immediately contiguous in the naturally occurring genome of an organism) that encodes an OCT-3 polypeptide. As used herein, an OCT-3 polypeptide is a polypeptide that: (1) is expressed in the plasma membrane of a biological cell (e.g., a cell in the kidney, liver, or brain), (2) contains transmembrane (TM) domains, and (3) when functioning normally, transports organic molecules across the plasma membrane. Preferably, the OCT-3 polypeptide has at least 6 transmembrane domains (e.g., 6, 8, or 10 TM domains), and more preferably, has at least 12 TM domains. The OCT-3 polypeptide can be a mammalian polypeptide, i.e., a polypeptide normally expressed by the cells of a mammal, such as a human. In the event the OCT-3 polypeptide is human, it can have the sequence shown in 2 or SEQ ID NO:4, or it can be encoded by nucleic acid molecules having the sequence shown in SEQ ID NO:1 or SEQ ID NO:3. However, the invention is not limited to nucleic acid molecules and polypeptides that are identical to those SEQ ID NOs. For example, the invention includes nucleic acid molecules which encode splice variants, allelic variants or mutant forms of OCT-3 as well as the proteins encoded by such nucleic acid molecules. Also within the invention are nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the sequence of SEQ ID NOs:1, 3, or 5. As described further below, molecules that are substantially identical to SEQ ID NOs:1-6 are also encompassed.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given compound (e.g., an OCT-3 polypeptide) means that the compound is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the compound may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). When chemically synthesized, a compound of the invention is substantially pure when it is substantially free from the chemical compounds used in the process of its synthesis. Polypeptides or other compounds of interest are substantially free from other compounds when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.
For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 100 nucleotides (e.g., 150, 200, 250, or 300 nucleotides).
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters as specified therein.
The invention also features a host cell that includes an isolated nucleic acid molecule encoding OCT-3 (either alone or in conjunction with a heterologous polypeptide, such as a detectable marker), or a nucleic acid vector that contains a sequence encoding OCT-3 (again, with or without a heterologous polypeptide). The vector can be an expression vector, and can include a regulatory element.
An antibody that specifically binds an OCT-3 polypeptide is also within the scope of the present invention and is useful, for example, to detect OCT-3 in a biological sample, or to alter the activity of OCT-3. For example, OCT-3 can be detected in a biological sample by contacting the sample with an antibody that specifically binds OCT-3 under conditions that allow the formation of an OCT-3-antibody complex and detecting the complex, if present, as an indication of the presence of OCT-3 in the sample. The use of an antibody in a treatment regime, where it can alter the activity of OCT-3, is discussed further below.
An antibody of the invention can be a monoclonal, polyclonal, or engineered antibody that specifically binds OCT-3 (as described more fully below). An antibody that xe2x80x9cspecifically bindsxe2x80x9d to a particular antigen, for example, an OCT-3 polypeptide of the invention, will not substantially recognize or bind to other molecules in a sample, such as a biological sample, that includes OCT-3.
Given that an object of the present invention is to alter the expression or activity of OCT-3 in vivo, a pharmaceutical composition containing, for example, an isolated nucleic acid molecule encoding OCT-3 (or a fragment thereof), a nucleic acid molecule that is antisense to OCT-3 (i.e., that has a sequence that is the reverse and complement of a portion of the coding strand of an OCT-3 gene), an OCT-3 polypeptide, or an antibody, small molecule, or other compound that specifically binds an OCT-3 polypeptide is also a feature of the invention.
The discovery and characterization of oct-3 and the polypeptide it encodes makes it possible to determine whether a given disorder is associated with aberrent expression of oct-3 (meaning expression at the level of gene transcription or mRNA translation) or activity of OCT-3. For example, one can diagnose a patient as having a disorder associated with aberrant expression of oct-3 by measuring oct-3 expression in a biological sample obtained from the patient. An increase or decrease in oct-3 expression in the biological sample, compared with oct-3 expression in a control sample (e.g., a sample of the same tissue collected from one or more healthy individuals) indicates that the patient has a disorder associated with aberrant expression of oct-3. Similarly, one can diagnose a patient as having a disorder associated with aberrant activity of OCT-3 by measuring OCT-3 activity in a biological sample obtained from the patient. An increase or decrease in OCT-3 activity in the biological sample as compared with OCT-3 activity in a control sample, indicates that the patient has a disorder associated with aberrant activity of OCT-3. The techniques required to measure gene expression or polypeptide activity are well known to those of ordinary skill in the art.
In addition to diagnostic methods, such as those described above, the present invention encompasses methods and compositions for typing and evaluating the prognosis, appropriate treatment, and treatment effectiveness of disorders associated with inappropriate expression of oct-3 or inappropriate activity of OCT-3. For example, the nucleic acid molecules of the invention can be used as probes to classify cells in terms of their level of oct-3 expression, or as primers for diagnostic PCR analysis in which mutations, allelic variations, and regulatory defects in the oct-3 gene can be detected. Similarly, those of ordinary skill in the art can use routine techniques to identify inappropriate activity of OCT-3, which can be observed in a variety of forms. For example, inappropriate activity can take the form of an alteration in the rate with which an OCT-3 transporter moves molecules across the plasma membrane, or a difference in the type of molecule that is transported. Diagnostic kits for the practice of such methods are also provided.
The invention further encompasses transgenic animals that express an oct-3 gene and recombinant xe2x80x9cknock-outxe2x80x9d animals that fail to express an oct-3 gene. These animals can serve as new and useful models of disorders in which oct-3 is misexpressed.
The invention also features antagonists and agonists of OCT-3 that can inhibit or stimulate, respectively, one or more of the biological activities of OCT-3. Suitable antagonists can include small molecules (i.e., organic or inorganic compounds with a molecular weight less than about 10,000 grams per mole), large molecules (i.e., molecules with a molecular weight greater than about 10,000 grams per mole), antibodies that specifically bind and xe2x80x9cneutralizexe2x80x9d OCT-3 (as described below), and nucleic acid molecules that interfere with transcription or translation of OCT-3 (e.g., antisense nucleic acid molecules and ribozymes). Agonists of OCT-3 also include small and large molecules, and antibodies other than neutralizing antibodies.
The invention also features molecules that can increase or decrease the expression of oct-3 (e.g., by altering transcription or translation). Small molecules (as defined above), large molecules (as defined above), and nucleic acid molecules (e.g., antisense and ribozyme molecules) can be used to inhibit the expression of an oct-3 gene. Other types of nucleic acid molecules (e.g., molecules that bind to oct-3 transcriptional regulatory sequences) can be used to increase the expression of an oct-3 gene.
Compounds that modulate the expression of oct-3 in a cell can be identified by comparing the level of expression of oct-3 in the presence of a selected compound with the level of expression of oct-3 in the absence of that compound. A difference in the level of oct-3 expression indicates that the selected compound modulates the expression of oct-3 in the cell. A comparable test for compounds that modulate the activity of OCT-3 can be carried out by comparing the level of OCT-3 activity in the presence and absence of the compound.
Patients who have a neurological disorder mediated by abnormal OCT-3 activity can be treated by administration of a compound that alters the expression of the oct-3 gene or the activity of the OCT-3 protein. When the objective is to decrease expression or activity, the compound administered can be an oct-3 antisense oligonucleotide, an antibody, such as a neutralizing antibody, or an antagonist that specifically binds oct-3 or OCT-3, respectively.
A wide variety of OCT-3 mediated neurological disorders are amenable to treatment according to the methods set forth herein. For example, the neurological disorder can be a neurodegenerative disease, such as Parkinson""s Disease, Huntington""s Disease, or Alzheimer""s Disease. Alternatively, the neurological disorder can be characterized by abnormal behavior or mood disorders. For example, the patient may suffer from depression, anxiety, or schizophrenia. A patient who has a sleep disorder or a weight disorder, such as a patient who is obese or who is suffering from a wasting disorder (as often occurs in the event of AIDS) can also be treated by administration of a compound that alters the expression of oct-3 or the activity of OCT-3. The neurological disorder can be pain, e.g., pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill), pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; and pain associated with surgery, rheumatoid arthritis, viral infection, allergic reaction, asthma, chronic pain, chronic pancreatitis, somatoform disorders, fibromyalgia syndrome; and neuropathic pain, allodynia, and hyperesthesia. A detailed description of these methods of treatment is set forth below.
The preferred methods and materials are described below in examples which are meant to illustrate, not limit, the invention. Skilled artisans will recognize methods and materials that are similar or equivalent to those described herein, and that can be used in the practice or testing of the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description and from the claims.